Method of isolating a peptide which immunologically mimics microbial carbohydrates including group B streptococcal carbohydrates and the use thereof in a vaccine

ABSTRACT

This invention relates to new vaccines against microorganisms based on antigenically mimetic peptides. The invention also relates to methods of discovering such mimetic peptides by first screening peptide-display phage libraries with antibodies against the microbial carbohydrates(s) of interest to locate antigenically mimetic peptides. Vaccines against Group B Streptococcus, or  Streptococcus Agalactiae , are preferably produced using this method.

I. STATEMENT OF RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/070,118, filed Dec. 31, 1997, which is herebyincorporated by reference.

II. FIELD OF THE INVENTION

[0002] This invention relates to new vaccines against microorganismsbased on antigenically mimetic peptides. The invention also relates tomethods of discovering such mimetic peptides by screeningpeptide-display phage libraries with antibodies against the microbialcarbohydrates(s) of interest to locate antigenically mimetic peptides.Vaccines against Group B Streptococcus, or Streptococcus Agalactiae, canbe produced using this method. Vaccines against other microbialpathogens may also be produced using this method.

III. BACKGROUND OF THE INVENTION

[0003] Vaccines protect against disease by harnessing the body's innateability to protect itself against foreign invading agents. Duringvaccination, the patient is injected with antigens, or DNA encodingantigens, which generate protective antibodies but which typicallycannot cause severe disease themselves. For example, vaccination forbacterial diseases such as typhoid fever consists of injecting a patientwith the bacterial agents of these diseases, after they have beendisabled in some fashion to prevent them from causing disease. Thepatient's body recognizes these bacteria nonetheless and generates anantibody response against them.

[0004] It is not always possible, however, to stimulate antibodyformation merely by injecting the foreign agent which causes thedisease. The foreign agent must be immunogenic, that is, it must be ableto induce an immune response. Certain agents such as tetanus toxoid areinnately immunogenic, and may be administered in vaccines. Otherclinically important agents are not immunogenic, however, and must beconverted into immunogenic molecules before they can induce an immuneresponse. Successfully accomplishing this conversion for a variety ofantigens is a major goal of a great deal of immunologic research.

[0005] However, researchers have yet to successfully convert a varietyof poorly immunogenic antigens into optimally immunogenic molecules. Ofparticular importance to the present invention is the failure ofimmunologic researchers to successfully convert carbohydrates intooptimally immunogenic molecules.

[0006] Carbohydrates are poorly immunogenic largely because of the wayin which they interact with the body's immune system. Carbohydratesfrequently function as T-independent antigens, which cannot be properlyprocessed by the antigen presenting cells that begin the typicalmammalian immune response. By contrast, T-dependent antigens areinitially processed by antigen presenting cells and then rely on T-cellsto stimulate B cells to manufacture large quantities of antibodiesagainst the antigen. As a result of these molecular biologicaldifferences, T-dependent antigens are immunologically superior toT-independent antigens, including carbohydrates, in three ways:

[0007] (1) T-dependent antigens are remembered by the immune systemwhile T-independent antigens are not. Thus, after vaccination, aninfection with a T-dependent antigen will be met with an extremely swiftand concentrated antibody attack compared to the response to the initialvaccination. Infections with T-independent antigens, by contrast,generally receive the same level of antibody response, even aftervaccination;

[0008] (2) T-dependent antigens are met with specific antibodies ofincreasing affinity against them over time, while T-independent antigensare met with antibodies of constant affinity; and

[0009] (3) T-dependent antigens stimulate a neonatal or immature immunesystem more effectively than T-independent antigens.

[0010] One approach which researchers have taken to enhance the immuneresponse to T-independent antigens is to inject subjects withpolysaccharide or oligosaccharide antigens that have been conjugated toa single T-dependent antigen such as tetanus or diphtheria toxoid.(Kasper, D., et al., J. Clin. Invest., Vol. 98, No. 10 2308-2314, 1996)(Schneerson, R. et al., Inf. Immun. 52:519, 1986) (Anderson, P W, etal., J. Immunol. 142:2464, 1989). These conjugate vaccines improve onvaccines based on carbohydrates alone because they “trick” the T-cellsinto directing the immune response, giving this response something ofthe character of a T-dependent response, even though it is directedagainst a T-dependent/T-independent conjugate. However, this “trick” isimperfect—although T-cells do assist, their assistance againstconjugates is not as effective as it is against true T-dependentantigens. As a result, generally only low levels of antibody titres areelicited, and only some subjects respond to initial immunizations. Thus,several immunizations are frequently required. This poses a seriousobstacle because patients are not always willing, or able, to completethis entire process; this is often true, for example, of patients wholive a great distance from medical facilities, as is frequently the casefor patients in lesser developed nations. And even when patients docomplete the process, there is no guarantee of success—infants less thantwo months of age may mount little or no antibody response even afterrepeated immunization. Furthermore, the process itself sometimes takesso long that patients contract the disease in a virulent form beforethey have been properly vaccinated.

[0011] In another attempt to gain the advantages of T-dependent responsewith T-independent antigens, including carbohydrates, researchers haveattempted to discover T-dependent antigens which are structurallyrelated to the T-independent antigen of interest. In theory, thesestructural mimics might elicit a superior immune response, compared to avaccine based on either the original T-independent antigen alone or aspart of a conjugate. Under this approach, at least, no part of theantigen in the vaccine is incompatible with T-cell assistance.

[0012] Yet locating T-dependent antigens which are sufficientlystructurally related to T-independent antigens to be true immunologicalmimics has proven difficult. Researchers have taken three differentapproaches to this problem, each of which has serious limitations.

[0013] First, some researchers have succeeded in designing syntheticpeptides which are immunologically mimetic by using computer simulationsand protein databases to construct a protein structure which closelyresembles the structure of the T-independent antigen of interest, asascertained through x-ray crystallography. (Westemik et al., Proc. Nat.Acad. Sci. USA Vol. 92, 4021-4025, 1995). However, this approach is onlyas good as the researcher's knowledge of the various structuresinvolved, which is frequently far from complete. Furthermore, becauseeven a single amino acid error can have a profound effect on theimmunogenicity of the synthetic peptide, as Westernik notes, a very highlevel of precision is required—higher than may be possible for molecularsystems whose structure is not well understood.

[0014] Second, some researchers have generated immunologic mimics byisolating anti-idiotypic antibodies which can elicit an immune responseto carbohydrate antigens of S. pneumonia (McNamara et al., Science226:1325, 1984), P. aeruginosa (Schreiber et al, J. Inf. Dis. 164:507,1991), E. coli (Kacack, M. B. et al., Infec. Immun. 61:2289, 1991) andGroup A Streptococci (Manafo, W. J. et al., J. Immunol. 139:2702, 1987).Anti-idiotypic antibodies are known to be structurally similar to theantigens of interest because of their design: they are generated againstthe idiotypes of antibodies which are known to specifically bind thecarbohydrate of interest. As a result, the anti-idiotypic antibody andthe carbohydrate bind specifically to the same idiotype structure (anantigenic determining structure in the antigen-binding portion of thecarbohydrate binding antibody). Thus, much as two keys which fit thesame lock have a high level of structural similarity, anti-idiotypicantibodies are thought to be structurally similar to theantibody-binding structures on carbohydrates. However, the similarity isnot complete: these are still antibodies, isolated from the cells ofmice, not complete carbohydrate structural mimics. As a result, therehas been some concern that, for treatment of humans, human vaccinesbased on anti-idiotypic antibodies would be undesirable because ofserious allergic reactions which could result. (Westernik, M. A. et al.,Proc. Nat. Acad. Sci. USA vol. 92, 4021, 1995.) This concern has led atleast some researchers to seek alternative means of discoveringT-dependent antigens which are structurally similar to T-independentantigens. (Westernik, M. A. et al., Proc. Nat. Acad. Sci. USA vol. 92,4021, 1995).

[0015] Finally, some researchers have sought to discover T-dependentantigens which are structurally similar to T-independent antigens byscreening libraries of phages, which express hundreds of millions ofrandom peptide sequences, using known carbohydrate-binding antibodies tofind particularly promising peptides. (Harris, S. et al., Proc. Natl.Acad. Sci. vol. 94, no. 6 pp. 2454-2459, 1997) (Valuation, P. et al., J.Mol. Biol. 261: 11-22, 1996) (Hoess, R. et al, Gene 128:43, 1993). (SeeOldenberg, K. R., Proc. Nat. Acad. Sci. USA 89:5393, 1992 (using lectinsto screen such libraries)). The approach outlined in these references issound only if one accepts that antigenic mimicry (meaning that thepeptide mimic binds the same highly specific antibody as thecarbohydrate of interest) is reasonably predictive of immunologicmimicry (meaning that the peptide will generate an immune responseagainst the carbohydrate of interest). After all, if antigenic mimicsare only rarely immunologic mimics, this procedure leaves one with farmore peptide sequence candidates for immunologic testing after theantigenic screening step than can reasonably be tested. Indeed, afterseveral failed attempts at obtaining an immunologic mimic using thisapproach were conducted, many in the art have in essence concluded thatthis approach is fundamentally flawed. In particular, at least oneresearcher has concluded that antigenic mimicry is rarely predictive oftrue immunologic mimicry, because the mechanism of peptide-antibodybinding is different than carbohydrate-antibody binding. (See Harris, S.et al., Proc. Natl. Acad. Sci. Vol. 94 No. 6 pp. 2454-2459, 1997).

[0016] Another serious limitation of both this approach and thedesign-approach of Westernik is that there is no a priori reason tobelieve that a peptide-based structural mimic necessarily exists for anygiven carbohydrate. The molecular basis underlying mimicry is unknown,and as such, offers no assurance that all carbohydrates structures havepeptide mimics. There is certainly evidence in nature that somecarbohydrate structures possess protein mimics. For example, the proteintendamistat is known to bind to the enzyme α-amylase at the samelocation this enzyme binds carbohydrates. And further research withsynthetic peptides has demonstrated a certain level of mimicry in avariety of carbohydrates drawn from a number of species, although thetheoretic basis for much of this data has been questioned. (See Harris,S. et al., Proc. Natl. Acad. Sci. vol. 94, no. 6 pp. 2454-2459, 1997).Nevertheless, from these studies, it appears that each new carbohydratepresents a unique challenge to this area of research.

[0017] Partly as a result of all of these limitations, there remains aneed in the art for vaccines effective against T-independent antigensand a method for developing such vaccines.

[0018] This need is particularly acute for vaccines effective againstGroup B Streptococci (GBS). Efforts at making a vaccine against GBS havefocused on using the T-independent polysaccharide of GBS. However, as isfrequently the case with T-independent antigens, vaccines containingonly GBS polysaccharides have been only marginally effective in inducingantibody. (Baker, C. J. et al., New Eng. J. Med. 319:1180). Conjugatevaccines containing the GBS polysaccharide conjugated to tetanus toxoid,a protein carrier, have been more successful. (Kasper, D. L. et al., J.Clin. Invest. 98:2308). Nevertheless, there is considerable room forimprovement in this area of the art.

[0019] This unmet need for novel vaccines against Group B Streptococci,or Streptococcus agalactiae, is only compounded by the widespread andfrequently deadly infections attributed to this bacterial agent. TheCenter for Disease Control has recently declared prevention of GBSinfections a major public health priority. (CDC, Morbidity and MortalityWeekly Report 45 (No. RR-7):1, 1996.) GBS causes invasive infections ofnewborns, pregnant women, and adults with underlying medical conditions.Although the bacteria are sensitive to antibiotics such as penicillin,case fatality rates are estimated to be 5-20% in newborn children and15-32% in adults. Infection is most commonly seen as bacteremia,meningitis, and pneumonia. Newborns who survive the disease may sufferpermanent neurologic sequelae as a result of meningitis. When motherslack protective anti-GBS antibodies, their newborn children are at riskof infection. (Baker, C. J. et al, New Eng. J. Med. 294:753, 1976)(Baker, C. J. et al., J. Infect. Dis. 136:598, 1977) (Hemming, V. G. etal., J. Clin. Invest. 58:1379, 1976). The development of maternalvaccines is considered a leading approach to the prevention of GBSdisease in newborns. (Mohle-Boetani, J. C. et al., J. Am. Med. Assoc.270:1442, 1993.) Passive administration of antibody has definedprotective epitopes of GBS (Pincus, S. H. et al., J. Immunol. 140:2779,1988) (Shigeoka, A. O. et al., J. Infect. Dis. 149:363, 1984) (Shigeoka,A. O. et al., Antibiot. Chemother. 35:254, 1985) (Egan, M. L. et al., J.Exp. Med. 158:1006, 1983) (Raff, H. V. et al., J. Exp. Med. 168:905,1988) (Lancefield, R. C. et al., J. Exp. Med. 142: 165, 1975), responsesto which are critical for an effective vaccine.

[0020] Thus, there remains in the art a need for improved vaccinesagainst GBS and methods for producing them.

IV. SUMMARY OF THE INVENTION

[0021] The present invention addresses this unmet need for novelvaccines by providing a method to isolate a peptide whichimmunologically mimics GBS. This is the first demonstration that apeptide structural mimic exists for this bacteria.

[0022] In addition, this invention meets a more general need in the artbecause it succeeds in locating mimics where the other two generalmethods for isolating immunological mimics would fail, or result insuboptimal mimics. First, this invention can succeed in locating animmunologic mimic even when the structure of the carbohydrate antigen isunknown or when an immunologic mimic of that structure cannot beconstructed from protein databases and computer simulations, unlike themethod of Westernik et al. Second, this invention ultimately results ina vaccine which does not have the same potential for serious allergicreaction possessed by anti-idiotypic antibody based vaccines.

[0023] The present invention also offers a validation of the finalmethod to isolating mimetic proteins by contradicting the findings ofHarris, et al. Harris concludes that the binding of peptides is by adifferent mechanism than binding of carbohydrate, and that this isneither antigenic nor immunologic mimicry. (Harris, S. et al., Proc.Natl. Acad. Sci. Vol. 94 No. 6 pp. 2454-2459, 1997.) Harris et al. basesthis conclusion on observations made when isolating peptides mimickinggroup A streptococcal cell-wall polysaccharide. Harris et al. observesthat most peptides isolated as potential antigenic mimics were primarilyreactive with the antibody used to isolated it, but only weakly reactivewith other antibodies against the same cell-wall polysaccharide.

[0024] However, the data set forth below contradicts this data, as wellas the conclusions drawn from it. In particular, it shows that two outof three IgG monoclonal antibodies to type III GBS also bind to thepeptide isolated by the method of the present invention. (FIG. 1b). Thisdata indicates that, while each of the monoclonal antibodies binds tothe same polysaccharide structure, some recognize different aspects ofthat structure. This interpretation was considered by Harris, et al. butdiscarded in favor of Harris's conclusion that the binding of peptidesis by a different mechanism than binding of carbohydrate. (Harris, S. etal., Proc. Natl. Acad. Sci. Vol. 94 No. 6, at 2459, 1997.) Moreover, thedata set forth below demonstrates that polyclonal anti-GBS antibodiesbind well to the peptide mimetic. Thus the present invention contradictsthe Harris findings.

[0025] The present invention relates to a method of isolating a peptidewhich immunologically mimics a portion of Group B streptococci,comprising the steps of:

[0026] (1) identifying protective antibodies reactive with said Group Bstreptococci;

[0027] (2) contacting a phage-display library, having phage, with one ormore of said protective antibodies identified in step 1;

[0028] (3) isolating one or more phage, having a displayed peptide,which bind one or more of the protective antibodies; and

[0029] (4) selecting, for all said phage isolated in step 3, thepeptides or peptide fragments to which the antibodies have bound.

[0030] Thus, the invention includes all mimetic peptides and peptidefragments that induce antibodies to GBS. Although any suitable portionof the GBS, such as lipotechoic acid or proteins, may be employed in theinvention, in a preferred embodiment, the mimetic peptides induceantibodies against the GBS carbohydrate. In another embodiment, themimetic peptide induces antibodies to the type III polysaccharide ofGBS. In a more preferred embodiment, the invention relates to peptideshaving the sequence FDTGAFDPDWPA (SEQ ID NO: 1) or FDTGAFDPDWPAC (SEQ IDNO:2) or WENWMMGNA (SEQ ID NO:3) or WENWMMGNAC (SEQ ID NO:4) andfragments and derivatives thereof which exhibit the same or similarability.

[0031] The present invention also relates to a vaccine consisting ofthese peptides and/or peptide fragments and/or derivatives together witha pharmaceutically acceptable carrier.

[0032] In yet another embodiment, the peptides and/or peptide fragmentsand/or derivatives of the invention may be conjugated to a carrier. In afurther embodiment, multiple copies of the peptide are used. In anotherembodiment, a fusion protein containing the peptide is employed. In yetanother embodiment, the vaccine uses DNA encoding for the peptide, theconjugate, or the fusion protein.

[0033] The present invention also relates method of treating a patient,comprising administering to the patient an immunostimulatory amount ofthe vaccine of the invention.

V. BRIEF DESCRIPTION OF THE DRAWINGS

[0034]FIG. 1 Binding of antibodies to synthetic peptide FDTGAFDPDWPAC(SEQ ID NO:2). Microtiter wells were coated with peptide at 10 μg/ml andthen blocked with 1% BSA. Test antibodies were added to the wells,incubated and washed. Antibody binding was detected with alkalinephosphatase conjugated anti-Ig and then substrate. The values are A₄₀₅,mean of duplicate or triplicate samples. The binding of monoclonalantibodies S7 and S9 is shown in panel A. Panel B shows the binding ofthree IgG anti-GBS type III and one irrelevant monoclonal antibodies,and panel C shows the serum from mice infected with 10⁸ live GBS typeIII (primary, secondary, and tertiary refer to the number of times themice were infected). The binding of all antibodies to BSA was <0.1.

[0035]FIG. 2 Competitive inhibition assays. Panel A shows inhibition ofS9 binding to GBS by peptide. Antibodies S7 or S9 were diluted to theconcentrations shown and mixed with the indicated concentration ofpeptide FDTGAFDPDWPAC (SEQ ID NO:2). Following a one hour incubation,the peptide and antibody were transferred to GBS-coated microtiter wellsand incubated overnight. The plates were washed and antibody binding wasdetected with alkaline phosphatase-conjugated anti-mouse IgM andsubstrate. The values are A₄₀₅, mean of duplicate samples. Panel B showsthe inhibition of anti-GBS antiserum (secondary bleed from panel 1C)binding to FDTGAFDPDWPAC (SEQ ID NO:2). The indicated dilutions of serumwere mixed with intact GBS or purified type III capsular polysaccharide(III-CPS) and plated into peptide-coated wells. The plates were washedand antibody binding was detected with alkaline phosphatase anti-mouseIg and substrate. The values are the mean and SEM of triplicate samples(if no error bars are seen, the SEM is too small to be drawn).

[0036]FIG. 3 Immunization of mice with peptide-carrier conjugatesresults in the production of GBS antibodies. Mice were immunized withpeptide FDTGAFDPDWPAC (SEQ ID NO:2) conjugated to OVA (mouse 1 and 2),KLH (mouse 3 and 4), or BSA (mouse 5 and 6), or with live GBS, once (1°)or twice (2°). Both prebleed and post-immune sera were diluted 1:1000and tested for binding to type III GBS (left panel) or to type IIIcapsular polysaccharide (right panel).

VI. DETAILED DESCRIPTION OF THE INVENTION

[0037] In developing the invention, the inventor used monoclonalantibody S9, a protective IgM monoclonal antibody against the type IIIcapsular polysaccharide (III-CPS) of Group B Streptococci (GBS) was used(Christensen, R. D. et al., Pediatr. Res. 17:795, 1983) (Pincus, S. H.et al, J. Immunol. 140:2779, 1988) (Shigeoka, A. O. et al, J. Infect.Dis. 149:363, 1984) to select epitope analogues from a peptide-displayphage library. The library consisted of millions of phage expressingdifferent fusion proteins from their surface. Depending upon desorptionconditions, two populations of phage were identified with displayedsequences of WENWMMGNA (SEQ ID NO:3) and FDTGAFDPDWPA.(SEQ ID NO:1)(Example 1.) Both sequences have aromatic, acidic, and hydrophobicresidues. The presence of aromatic residues is characteristically seenin peptides mimicking carbohydrates. (See Westemik, M. A. et al, Proc.Natl. Acad. Sci. USA 92:4021 at 4025, 1995.) The presence of acidicresidues probably reflects the sialic acid in the carbohydrate epitope.However the molecular basis underlying the antigenic mimicry of thecarbohydrate structure by the peptides is not known and this descriptionis not intended to limit the invention.

[0038] As the study progressed, ELISA results demonstrated that phagewith these two displayed sequences bound to S9 and no other antibodies.(Example 2.) Phage blocked the binding of S9 to type III GBS, but didnot block the binding of another anti-GBS monoclonal antibody. (Example3.) Phage displaying the FDTGAFDPDWPA (SEQ ID NO: 1) sequence showedgreater inhibition. (Example 3.) Antibody S9 and other monoclonal andpolyclonal anti-GBS type III antisera bound the synthetic peptideFDTGAFDPDWPAC (SEQ ID NO:2). (Example 3.) The binding of S9 to GBS wasinhibited by the free peptide with an IC₅₀ of 30 μg/ml. (Example 3.) Thebinding of polyclonal anti-GBS antibodies to peptide could be blocked byintact GBS, as well as by purified capsular polysaccharide. (Example 4.)The peptide was conjugated to three different carriers and used toimmunize mice. (Example 5.) All mice produced a significant antibodyresponse to GBS and to the purified capsular polysaccharide following asingle immunization. (Example 5.) These data demonstrate that a peptidemimetic of the GBS capsular polysaccharide is both antigenic andimmunogenic. Such peptides will provide more efficacious vaccinepreparations against important carbohydrate epitopes.

[0039] Thus, the present invention relates to a method of isolating apeptide which immunologically mimics a portion of Group B streptococci,comprising the steps of:

[0040] (1) identifying protective antibodies reactive with said Group Bstreptococci;

[0041] (2) contacting a phage-display library, having phage, with one ormore of said protective antibodies identified in step 1;

[0042] (3) isolating one or more phage, having a displayed peptide,which bind one or more of the protective antibodies; and

[0043] (4) selecting, for all said phage isolated in step 3, thepeptides or peptide fragments to which the antibodies have bound.

[0044] A peptide which “immunologically mimics” GBS is a substance thatelicits an antibody response against the GBS. The peptide is preferablygreater than five amino acids, although peptides of any length arewithin the scope of the invention.

[0045] The protective antibodies within the invention are antibodiesshown to protect against GBS infection or to ameliorate the effects of aGBS infection. Any bactericidal assay that is known in the art may beused to identify the protective antibodies of the invention, althoughthe assay set forth in U.S. Pat. No. 5,971,511, is preferred. Inaddition, protective antibodies can be identified by use of the lethalchallenge assay in which laboratory animals, generally mice, areinjected with a lethal amount of the bacteria being tested. Antibodiesare then administered and mouse survival is determined. Those antibodiesthat are able to protect against death are considered to be protective.

[0046] “Contacting”, as used herein, refers to incubation for asufficient period of time to permit antibody/antigen binding to occur,as can be easily measured by methods routine in the art.

[0047] “Phage display library”, as used herein, refers to a multiplicityof phage which express random amino acid sequences of between 7 and 15amino acids at a location which may be bound by an antibody.

[0048] Particularly preferred in the practice of this invention is aphage display library produced by the method of Burritt et al. (Burritt,J. B. et al., Analyt. Biochem. 338:1). Phage has the usual meaning it isgiven by one of ordinary skill in the art. (See, for example, Maniatis,et al., Molecular cloning: a laboratory manual. Cold Spring Harbor(1982)) The phrase “antibodies specific for GBS” refers to monoclonal orpolyclonal antibodies which bind the substance GBS with an affinitygreater than the average unselective affinity which these antibodiesshow for substances structurally unrelated to GBS. Although antibodiesof all isotypes, i.e., IgG, IgA, IgM, IgD, and IgE, may be used, in oneembodiment, IgM antibodies are preferred.

[0049] A particularly preferred antibody in the practice of thisinvention is monoclonal antibody S9, a protective monoclonal antibody ofthe IgM isotype against the type III capsular polysaccharide of group BStreptococci.

[0050] As noted above, any portion of the Group B streptococci, such aslipotechoic acid or proteins, may be used but the preferred portion isthe carbohydrate. A preferred carbohydrate is the capsularpolysaccharide, and a particularly preferred capsular polysaccharide isthe Type III capsular polysaccharide (associated with Type III Group BStreptococcus), although Type I III Type II are within the scope of theinvention.

[0051] The phrase “isolating one or more phage . . . which bind one ormore of the antibodies” means physically removing from the phage displaylibrary phage that bind the antibody with greater affinity than thatbetween the antibody and a structurally unrelated antigen.

[0052] Although any technique for isolation may be used in the practiceof the invention, a preferred method is affinity purification, which iswell known in the art. A particularly preferred means of isolating phagefrom the phage display library is to first preabsorb the libraryrepeatedly with cyanogen bromide activated sepharose 4B beads coatedwith antibodies specific for an antigen structurally unrelated to GroupB Streptococcus capsular polysaccharide, most preferably S10 antibody.Following repeated preabsorption, the library is incubated overnightwith cyanogen bromide activated sepharose 4B beads coated with one ormore antibodies specific for Group B Streptococcus. Following thisincubation, these same beads are washed extensively, and then elutedinto two separate pools, the first pool being formed by elution with0.1M glycine pH 2.2, with the second being formed by elution with 0.5MNH₄OH pH 7. Next, the eluated pools are amplified, most preferably in E.coli strain K91. Following elution, each pool is reapplied to thecolumn, and the same incubation and washing procedure employed, eluting(in the end) with the same substance used to generate the original pool.This process is repeated for each pool, most preferably three times.Finally, phage are tested for binding to the selecting antibody usingimmunoblots of plaques. Phage which pass this final round are cloned,amplified to high titer, and purified by precipitation, most preferablywith 2.5% polyethylene glycol.

[0053] “Displayed peptide” refers to peptides having an amino-acidsequence of between 7 and 15 amino acids which varies randomly betweeneach of the individual phage which make up the phage display library.

[0054] As noted above, the mimetic peptides obtained by this method mayinduce antibodies to any portion of the GBS, but preferably to acarbohydrate portion. Even more preferable are peptides that induceantibodies to capsular polysaccharides of GBS, including Type III.Specific peptides within the invention are FDTGAFDPDWPA (SEQ ID NO: 1)or FDTGAFDPDWPAC (SEQ ID NO:2) or WENWMMGNA (SEQ ID NO:3) or WENWMMGNAC(SEQ ID NO:4), but the invention also includes fragments and derivativesthereof that exhibit the same or similar activity.

[0055] Once the peptides are isolated by this process, they may be usedin a vaccine. The vaccine may include the peptide itself or the peptidemay be conjugated to a carrier or otherwise compounded. Carrierpreferably refers to a T-dependent antigen which can activate andrecruit T cells and thereby augment T-cell dependent antibodyproduction. However, the carrier need not be strongly immunogenic byitself, although strongly immunogenic carriers are within the scope ofthis invention. Multiple copies of the carrier are also within the scopeof this invention. Multiple copies of the peptide are also within thescope of the invention, either unconjugated or conjugated to one or morecopies of the carrier. Fragments and derivatives of the peptide are alsowithin the scope of the invention, either alone or in combination witheach other, not necessarily identically reproduced, and eitherunconjugated or conjugated to one or more copies of the carrier. Fusionproteins containing single or multiple copies of the peptide or partsthereof are also within the scope of the invention. In a furtherembodiment, microbes that express the DNA of the fusion protein arewithin the invention. In yet another embodiment, DNA encoding any andall of these substances is within the scope of the invention.

[0056] In a preferred embodiment, the carrier is a protein, a peptide, aT cell adjuvant or any other compound capable of enhancing the immuneresponse. The protein may be selected from a group consisting of but notlimited to viral, bacterial, parasitic, animal and fungal proteins. In amore preferred embodiment, the carrier is albumin (such as bovine serumalbumin (BSA)), keyhole limpet hemocyanin (KLH), ovalbumin (OVA),tetanus toxoid, diphtheria toxoid, or bacterial outer membrane protein,all of which may be obtained from biochemical or pharmaceutical supplycompanies or prepared by standard methodology (Cruse, JM (ed.) ConjugateVaccines in Contributions to Microbiology and Immunology vol. 10(1989)). Other proteins that could function as carriers would be knownto those of ordinary skill in the art of immunology.

[0057] The isolated peptides, with or without further compounding, maybe immunogenic or, alternatively, the immunogenicity may arise from thecompounding. Methods of measuring immunogenicity are well known to thosein the art and primarily include measurement of serum antibody includingmeasurement of amount, avidity, and isotype distribution at varioustimes after injection of the construct. Greater immunogenicity may bereflected by a higher titer and/or increased life span of theantibodies. Immunogenicity may also be measured by the ability to induceprotection to challenge with noxious substance or organisms.Immunogenicity may also be measured using in vitro bactericidal assaysas well as by the ability to immunize neonatal and/or immune defectivemice. Immunogenicity may be measured in the patient population to betreated or in a population that mimics the immune response of thepatient population.

[0058] A particularly preferred means of determining the immunogenicityof a given substance is to first obtain sera of mice both before andafter immunization with the substance. Following this, the strength ofthe post-immunization sera binding to GBS and capsular polysaccharide isascertained using an ELISA, and compared against the ELISA resultsobtained for the pre-immunization sera.

[0059] The peptides of the invention as well as the vaccines of theinvention may exhibit enhanced immunogenicity. Enhanced immunogenecityrefers to immunogenicity greater than that obtained by the Group BStreptococcal carbohydrate of interest alone, and preferably thatsufficient to effect a statistically measurable immunoprotective effect.As a point of reference, a peptide would certainly have enhancedimmunogenicity if it provoked a level of immunogenic response equal toor greater than that obtained by administration of purifiedcarbohydrate. In a preferred embodiment, the enhanced immunogenicity, asmeasured by an ELISA, is greater than that provoked by 10⁸ GBS, andcomparable to that seen after two injections with live GBS.

[0060] Pharmaceutically acceptable carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceutical carriersare described in Martin, E.W., Remington's Pharmaceutical Sciences,specifically incorporated herein by reference. These carriers can alsocontain immunoadjuvants, including but not limited to alum, aluminumcompounds (phosphate and hydroxide), and muramyl dipeptide derivatives.

[0061] The invention also relates to the treatment of a patient byadministration of an immunostimulatory amount of the vaccine. Patientrefers to any subject for whom the treatment may be beneficial andincludes mammals, especially humans, horses, cows, dogs, and cats aswell as other animals, such as chicks. An immunostimulatory amountrefers to that amount of vaccine that is able to stimulate the immuneresponse of the patient for the prevention, amelioration, or treatmentof diseases. Of course, as noted above, the immunostimulation may resultfrom the form of the antibody or the adjuvant with which it iscompounded.

[0062] The vaccine of the invention may be administered by any route,but is preferably administered topically, mucosally or orally. Othermethods of administration will be familiar to those of ordinary skill inthe art, including intravenous, intramuscular, intraperitoneal,intracoporeal, intrarticular, intrathecal, intravaginal, intranasal,oral and subcutaneous injections.

[0063] All references cited in the specification are hereby explicitlyincorporated by reference, even if no language in the citation itself soindicates.

METHODS

[0064] I. Antibodies, Bacteria, Phage Library and Peptide.

[0065] The murine monoclonal antibodies used in this study are listed inTable I, below. Antibodies S7, S9, and S10 are IgM antibodies againstGBS (Pincus, S. H. et al., J. Immunol. 140:2779), B6.1 is a protectiveIgM antibody directed against a β-1,2-linked trimannose epitope ofCandida albicans, (Han, Y. et al., Infect. Immun. 63:2714), and 924 isan irrelevant IgGi anti-HIV gp120 (Pincus, S. H. et al., J. Immunol.146:4315). Antibodies 1A6, 1B1, and 2A6 were produced by immunizationwith tetanus toxoid-type III capsular polysaccharide conjugate.(Jennings, H. J., manuscript in preparation). Polyclonal anti-type IIIGBS antiserum was obtained by repeated infection of BALB/c mice with 10⁸live GBS strain 1.2 as described elsewhere (Pincus, S. H. et al. Infect.Immun. 61:3761). Mice were bled 18 days following the first infection(primary bleed) and one week following the second and third infections.Rabbit antiserum against M13 bacteriophage was made in our laboratories.Alkaline phosphatase-conjugated anti-mouse IgG and anti-rabbit IgG werefrom Zymed Laboratories (South San Francisco, Calif.). GBS type III,strain 1.2, are described elsewhere. (Pincus, S. H. et al., J.Bacteriol. 174:3739). Capsular polysaccharide was extracted from GBSstrain 1.2 using a modification of the protocol of Lancefield(Lancefield, R. C. J. Hyg. (Lond.) 64:191). GBS were washed twice inwater, boiled in HCl pH 2.0 for 10 minutes, and the GBS pelleted out.The HCl extract was neutralized with tris base, chilled and precipitatedwith 80% ethanol. The ethanol extract was then treated with DNAase (5μg/ml), RNAase (1 μg/ml), and then proteinase k (200 μg/ml). The extractwas bound by antibody S9, but not by S7 indicating the presence of typeIII capsular antigen, but not group B carbohydrate. The phage displaylibrary utilized in these studies was produced by Dr. J. Burritt in thefilamentous phage M13 KBst and express a random 9AA peptide sequence asan amino-terminal fusion with the minor coat protein pIII. (Burritt, J.B. et al., Analyt. Biochem 338:1) (Deleo, F. R. et al., Proc. Nat. Acad.Sci. USA. 92:7110) (Smith, G. P. et al, Methods in Enzymology 217:228).The library has a complexity of 5×10⁸ unique phage. The amino acidsequence of the displayed peptide was derived by sequencing the chimericpIII-peptide gene utilizing automated sequencing methodology (Universityof Montana Molecular Biology Center, Missoula, Mont.). A syntheticpeptide of sequence FDTGAFDPDWPAC (SEQ ID NO:2) was made byBio-Synthesis (Lewisville, Tex.) using standard F-moc solid phasesynthesis protocols and was >70% pure by HPLC and mass spectrometeranalysis. TABLE I Antibodies Name Class Specificity Reference S7 IgMgroup B carbohydrate, all GBS (Pincus, S. H. et al., J. Immunol.140:2779, 1988) S9 IgM type III capsular polysaccharide (Pincus, S. H.,et al., J. Immunol. 140:2779, 1988) S10 IgM Beta-C protein of type I andII GBS (Pincus, S. H., et al., J. Immunol. 140:2779, 1988) 1A6 IgG1 typeIII capsular polysaccharide (Jennings, H. J., manuscript) 1B1 IgG2a typeIII capsular polysaccharide (Jennings, H. J., manuscript) 2A6 IgG1 typeIII capsular polysaccharide (Jennings, H. J., manuscript) 924 IgG1 HIVgp120 (Pincus, S. H., et al., J. Immunol. 146:4315, 1991) B6.1 IgMCandida albicans (Han, Y., et al., Infect. Immun. 63:2714, 1995) T6 IgG1Synthetic polypeptide (Y,E)-A--K (Pincus, S. H., et al., Mol. Immunol.19:1551, 1982)

[0066] II. Selection of Phage

[0067] Monoclonal antibodies S7, S9, and S10 were separately immobilizedon cyanogen bromide activated sepharose 4B (Sigma Chemical, St. Louis,Mo.) at 3 mg of antibody per ml of beads. Phage (4×10¹² plaque formingunits, pfu) were preadsorbed five times on beads containing immobilizedantibody S10 to remove any phage that would bind to all IgM antibodies.The preadsorbed library was divided into two aliquots, for affinityselection with either antibody S7 or S9. The phage, diluted in trisbuffered saline (TBS), 1% bovine serum albumin (BSA), and 1% Tween-20,were incubated overnight with 2.5 ml of immobilized antibody. The beadswere washed extensively with TBS/Tween (15 batchwise elutions of 15 mleach and on a column with 75 ml) and eluted with 0.1 M glycine pH 2.2.Following an additional wash, the beads were further eluted with 0.5 MNH₄OH pH 11. The eluted phage were neutralized to pH 7 immediately, andthose phage eluted with high or low pH were maintained as distinctpools. The titer of phage in the last wash and each eluate wasdetermined. The eluted phage were then amplified in E. coli strain K91to a titer of 1012 pfu and reapplied to the column. The same incubationand washing procedures were used, and bound phage eluted with eitherglycine or NH₄OH, depending upon which pool of phage was used. Eachaliquot of phage was subjected to three such rounds of selection. Thethird round eluate had >10⁸ pfu. Phage were tested for binding to theselecting antibody, but not to irrelevant IgMs using immunoblots ofplaques. Phage with the desired reactivity were cloned, amplified tohigh titer, and purified by precipitation with 2.5% polyethylene glycol(8000 MW), 0.5M NaCl.

[0068] III. ELISA

[0069] ELISA was used to measure the binding of phage to antibody,antibody to peptide, and antibody to GBS or capsular polysaccharide.Protein or peptide antigens were coated onto microtiter wells (Immulon2, Dynatech, McLean, Va.) at 5-10 μg/ml. GBS were coated onto microtiterwells using poly-1-lysine and glutaraldehyde as described elsewhere.(Pincus, S. H. et al., J. Immunol. 140:2779) (Pincus, S. H. et al.,Infect. Immun. 61:3761). Capsular polysaccharide was coated directlyonto microtiter wells. Plates were blocked with 1% BSA or 1% ovalbumin(OVA) and used within one week. Primary antibodies were incubated inmicrotiter wells at 4° for 18 hours. The plates were washed andincubated with alkaline phosphatase-conjugated anti-Ig for 6 hours,followed by washing and addition of colorimetric substrate p-nitrophenylphosphate (Sigma Chemical). A₄₀₅ was determined using a microplatereader (EL-320, Bio-Tek Instruments, Winooski, Vt.). Binding of phagewas measured by incubating phage in coated microtiter wells, washing,addition of rabbit anti-phage antiserum, and detection of rabbit Ig withalkaline phosphatase-conjugated anti-rabbit Ig.

[0070] IV. Immunization of Mice

[0071] Peptide was conjugated to maleimide-derivitized BSA, OVA, andkeyhole limpet hemocyanin (KLH, Pierce Chemical, Rockford, Ill.).Efficacy ofconjugation was demonstrated by reactivity of the conjugate,but not the unconjugated maleimide derivative, with antibody S9. Threegroups of two mice were immunized subcutaneously with a single 50 μgdose of each conjugate in complete Freund's adjuvant (DIFCO, DetroitMich.). Mice were bled on the day of immunization and day 35.

EXAMPLE 1 Selection of Phage

[0072] A phage-display library expressing a random nine AA sequence wasselected for binding to one of two different anti-GBS monoclonalantibodies: S9, a protective monoclonal antibody that binds to the typeIII capsular polysaccharide, and S7, specific for the group Bcarbohydrate. (Pincus, S. H. et al., J. Immunol. 140:2779). The latterwas used primarily as a specificity control. Within each selection, twoseparate desorption protocols were used to identify two populations ofphage: 0.1 M glycine pH 2.2 or 0.5M NH₄OH pH 11. Phage with bindingspecificity for the selecting antibody were identified by immunoblots ofplaques. Forty clones were selected, ten from each elution condition andselecting antibody, and amplified to a titer of 10¹³-10¹⁴ pfu/ml.

[0073] The DNA encoding the displayed peptide from the two differentpools of S9-selected phage was sequenced. Within each pool, the sequenceof each clone was identical, but two very different sequences were seendepending upon the eluting pH. The 9AA displayed sequence for theglycine pH 2.2 eluted phage was WENWMMGNA (SEQ ID NO:3). The sequencedisplayed by the NH₄OH-eluted phage was 12 AA long because there was asingle base deletion following the sequence encoding the displayedpeptide followed 8 bases later by a compensatory single base addition.The 12 AA sequence displayed by the NH₄OH-eluted phage was FDTGAFDPDWPA(SEQ ID NO: 1). Although these two sequences are considerably different,there are similarities in motif, in each case there are aromatic, acidicand hydrophobic residues.

EXAMPLE 2 Specificity of Antibody Binding to Displayed Peptides

[0074] To show the specificity of phage binding, ELISA plates werecoated with antibodies. The immobilized antibodies were incubated withrepresentative phage clones from each selection (10¹⁰ pfu per well) andbinding measured. The results are shown in Table II, below. The parentalphage (M13 KBst) bound to no antibody. Phage selected with antibody S7or S9 bound only to the selecting antibody. Phage that were firstabsorbed on antibody S10, prior to the selection on S7 or S9, bound toall IgM antibodies, indicating that within the library there is apopulation of phage that bind to all IgMs. TABLE II Binding of Phage toImmobilized Antibodies. ANTIBODY Phage Selection None 924 S7 S9 B6.1M13KBst none 0.05 0.12 0.12 0.08 0.16 S10-4 S10 0.08 0.13 1.62 1.54 2.41S10-8 S10 0.09 0.22 1.84 1.88 2.42 S9-11 S9-pH 2.2 0.05 0.16 0.10 1.220.17 S9-16 S9-pH 2.2 0.19 ND 0.18 1.15 0.10 S9-26 S9-pH 11 0.06 ND 0.151.80 0.07 S9-24 S9-pH 11 0.03 0.07 0.07 1.76 0.15 S7-A S7-pH 11 0.050.10 1.74 0.16 0.20 S7-B S7-pH 2.2 0.08 ND 1.91 0.28 0.49 # The valuesare A₄₀₅, mean of duplicate samples.

[0075] Binding of antibodies to the synthetic peptide FDTGAFDPDWPAC (SEQID NO:2) was also demonstrated by ELISA. FIG. 1 shows the binding of theantibodies to peptide. Panel A shows binding of the monoclonalantibodies S7 and S9. Binding to the peptide was seen only with antibodyS9. Panel B shows that two of three other IgG anti-GBS type IIImonoclonal antibodies bind to the peptide. Panel C shows that the seraof mice infected with type III GBS bind to the peptide; binding to thepeptide parallels the total antibody response to type III GBS (Pincus,S. H. et al., Infect. Immun. 61:3761). These data demonstrate thatanti-GBS antibodies other than the selecting antibody also recognize thepeptide sequence.

EXAMPLE 3 Phage and Peptides Block the Binding of Antibody to GBS

[0076] The demonstration of specific recognition of the peptide sequenceby the selecting antibody is a good indication that the phage bind tothe variable regions. However to demonstrate that the displayed sequenceactually resembles the carbohydrate epitope of GBS, blocking of antibodybinding to GBS antigens must be shown. To perform these experiments,ELISA was used where antibody and inhibitor (phage or peptide) werepremixed, incubated for one hour, and then plated onto the microtiterplates with GBS. Inhibition of the antibody's binding to GBS indicatedthat the phage or peptide was successfully competing with the GBS forantibody binding.

[0077] In Table III, below, intact phage were used to inhibit thebinding of antibodies S7 and S9 to GBS. The concentrations of S7 usedwere slightly higher than those of S9 because there are fewer antigenicdeterminants recognized by S7 on the surface of GBS. (Pincus, S. H. etal., J. Immunol. 140:2779). The concentrations of antibody used forinhibition are in middle third of the linear portion of the bindingcurve. Antibody B6.1 was used to indicate the level of backgroundbinding of IgM to GBS. The S9-selected phage inhibited the binding of S9but not S7 to GBS, while the S7-selected phage inhibited the binding ofonly S7. The parental phage did not produce significant inhibition ofeither S7 or S9. In some cases, the inhibition of antibody binding wasvirtually complete. The inhibition of S9 induced by the phage eluted athigh pH (S9-24 and S9-26) was considerably greater than that seen withthe phage eluted at low pH. To confirm that the phage eluted at high pHwere better inhibitors, a titration of phage was performed. (Table IV).The data indicate that the phage eluted at high pH were approximatelyfive times more efficient at inhibiting antibody S9 as the low pH phage;equivalent inhibition was seen with 2×10¹⁰ pfu S9-24 or S9-26 as with10¹¹ pfu S9-11 or S9-16. The greater inhibition may be a reflection ofthe AA sequence of the displayed peptide or of its greater length. Theincreased inhibition is an indication that the peptide displayed byphage clones S9-24 and S9-26 binds to antibody S9 with a higher aviditythan the other displayed peptides. TABLE III Inhibition by phage ofantibody binding to GBS S9 (μg/mI) S7 (μg/mI) Inhibitor 0.1 0.03 0.3 0.1no phage 2.03 0.63 2.25 0.93 M13KBst 1.97 0.51 2.04 0.97 S9-11 1.47 0.162.41 1.07 S9-16 0.89 0.14 1.94 1.10 S9-24 0.15 0.13 2.23 1.17 S9-26 0.210.14 2.47 1.02 S7-A 1.96 0.55 0.48 0.30 # Background binding ofirrelevant IgM antibody B6.1 is 0.14.

[0078] TABLE IV Titration of phage-mediated inhibition of antibodybinding to GBS Phase Number Ab Binding None 0 2.03 M13KBst 10¹¹ 1.97S9-11 10¹¹ 1.47 2 × 10¹⁰ 1.66 S9-16 10¹¹ 0.89 2 × 10¹⁰ 1.54 S9-24 10¹¹0.15 2 × 10¹⁰ 1.03 S9-26 10¹¹ 0.21 2 × 10¹⁰ 1.34

[0079] Because inhibition of antibody binding seen with intact phage mayhave resulted from steric inhibition due to the large size of afilamentous phage, the inhibition experiments were repeated with thesynthetic peptide FDTGAFDPDWPAC (SEQ ID NO:2). Those results are shownin FIG. 2, panel A. The binding of S9 to GBS was inhibited by freepeptide with an IC₅₀ of approximately 30 μg/ml. There was no inhibitionof the binding of antibody S7.

EXAMPLE 4 Binding of Anti-GBS Antibody to Peptide is Inhibited byCapsular Polysaccharide

[0080] To demonstrate that anti-GBS polyclonal antibodies which bind tothe peptide are specific for the type III capsular polysaccharide, bothintact GBS and extracted capsular polysaccharide were used to inhibitbinding to peptide (FIG. 2B). Two different dilutions of sera from miceinfected with GBS were premixed with either an equal volume of GBS (OD0.9) or dilutions of the capsular polysaccharide. The GBS gave completeinhibition of binding to peptide. Although the inhibition by thecapsular polysaccharide was not quite complete, the increasinginhibition with greater concentrations of polysaccharide or lesseramounts of serum suggest that the maximal inhibition had not beenobtained.

EXAMPLE 5 Mice Immunized with Peptide make Anti-GBS Antibody

[0081] Mice were immunized with peptide FDTGAFDPDWPAC (SEQ ID NO:2)conjugated to OVA (mouse 1 and 2), KLH (mouse 3 and 4), or BSA (mouse 5and 6), or with live GBS, once (1° ) or twice (2°). Conjugation can beaccomplished using standard methodology. (See Cruse, JM (ed.) ConjugateVaccines in Contributions to Microbiology and Immunology vol. 10(1989)). Both prebleed and post-immune sera were diluted 1:1000 andtested for binding to type III GBS or to type III capsularpolysaccharide. All mice made peptide-specific antibody as well asantibody to GBS and to purified capsular polysaccharide as shown in FIG.3. The left panel shows binding to type III GBS and the right panelshows binding to type III capsular polysaccharide. The resultsdemonstrate a high background of binding to GBS in the prebleed sera,perhaps as a result of binding to bacterial Fc receptors. As acomparison, the anti-GBS antibody response of mice that were infectedwith live GBS was also measured. A single immunization withpeptide-protein conjugate induced a greater anti-GBS antibody responsethan seen following infection with 10⁸ GBS and a response comparable tothat seen following a second infection.

[0082] Other embodiments of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. It is intended that the specificationand examples be considered as exemplary only, with a true scope andspirit of the invention being indicated by the following claims.

1 4 1 12 PRT Artificial Sequence Description of Artificial SequenceMimetic Peptide 1 Phe Asp Thr Gly Ala Phe Asp Pro Asp Trp Pro Ala 1 5 102 13 PRT Artificial Sequence Description of Artificial Sequence MimeticPeptide 2 Phe Asp Thr Gly Ala Phe Asp Pro Asp Trp Pro Ala Cys 1 5 10 3 9PRT Artificial Sequence Description of Artificial Sequence MimeticPeptide 3 Trp Glu Asn Trp Met Met Gly Asn Ala 1 5 4 10 PRT ArtificialSequence Description of Artificial Sequence Mimetic Peptide 4 Trp GluAsn Trp Met Met Gly Asn Ala Cys 1 5 10

I claim:
 1. A method of isolating a peptide which immunologically mimicsa portion of a Group B streptococci, comprising the steps of: (1)identifying protective antibodies reactive with said Group Bstreptococci; (2) contacting a phage-display library, having phage, withone or more of said protective antibodies identified in step 1; (3)isolating one or more phage, having a displayed peptide, which binds oneor more of the protective antibodies; and (4) selecting, for all saidphage isolated in step 3, the peptides or peptide fragments to which theantibodies have bound.
 2. The method of claim 1 wherein the portion ofthe Group B Streptococci is a carbohydrate.
 3. The method of claim 2,wherein the carbohydrate is a Type III capsular polysaccharide.
 4. Amimetic peptide that induces antibodies against a portion of a Group Bstreptococci.
 5. The peptide of claim 4 wherein the portion of the GroupB Streptococci is a carbohydrate.
 6. The peptide of claim 5 wherein thecarbohydrate is a Type III capsular polysaccharide.
 7. The peptide ofclaim 4 having a sequence selected from the group consisting ofFDTGAFDPDWPA (SEQ ID NO: 1), FDTGAFDPDWPAC (SEQ ID NO:2), WENWMMGNA (SEQID NO:3), and WENWMMGNAC (SEQ ID NO:4).
 8. A vaccine, comprising: amimetic peptide that induces antibodies against a portion of a Group Bstreptococci and a pharmaceutically acceptable carrier.
 9. The vaccineof claim 8 wherein the portion of the Group B Streptococci is acarbohydrate.
 10. The vaccine of claim 9 wherein the carbohydrate is aType III capsular polysaccharide.
 11. The vaccine of claim 8 wherein themimetic peptide has a sequence selected from the group consisting ofFDTGAFDPDWPA (SEQ ID NO:1), FDTGAFDPDWPAC (SEQ ID NO:2), WENWMMGNA (SEQID NO:3), and WENWMMGNAC (SEQ ID NO:4),
 12. A method of treating apatient, comprising administering to the patient an immunostimulatoryamount of a vaccine, comprising: a mimetic peptide that inducesantibodies against a portion of a Group B streptococci and apharmaceutically acceptable carrier.
 13. The method of claim 12 whereinthe portion of the Group B Streptococci is a carbohydrate.
 14. Themethod of claim 13 wherein the carbohydrate is a Type III capsularpolysaccharide.
 15. The method of claim 12 wherein the mimetic peptidehas a sequence selected from the group consisting of FDTGAFDPDWPA (SEQID NO:1), FDTGAFDPDWPAC (SEQ ID NO:2), WENWMMGNA (SEQ ID NO:3), andWENWMMGNAC (SEQ ID NO:4)